Abstract - Project 1: Natural Killer (NK) cells are a key component of anti-tumor immunity that are characterized by their ability to recognize and kill tumor targets in an antigen- and MHC-unrestricted manner, and elicit a pro-inflammatory environment that primes adaptive immunity. To overcome the low number and function of NK cells caused by cancer and its treatment, we developed a robust approach to propagate large numbers of NK cells for adoptive immunotherapy that relies on inducing a STAT3-mediated expression program. TGF? is a key factor that limits anti-tumor NK cell activity. Tumor profiling demonstrates high expression of TGF?-family genes in pediatric brain tumors and sarcomas. We show that the addition of TGF? during expansion epigenetically reprograms NK cells with novel functional characteristics, including TGF? resistance through SMAD3 suppression, hypersecretion of inflammatory cytokines, increased death ligands, and remodeling of gene expression patterns. AHR is a critical transcription factor involved in NK cell functional maturation, but also mediates inhibition through sensing of tryptophan (Trp) metabolites produced by enzymes IDO and TDO. Lastly, the biodistribution and homing of adoptively transferred NK cells is critical for their anti-tumor effect in solid tumors. We recently modified our propagation method to generate TGF?-resistant NK cells with high CXCR3 expression, and developed a novel approach for genetic modification of expanded NK cells using Cas9/ RNP complexes, enabling deletion of deleterious genes and targeted introduction of transgenes that may further improve number and function. Our overarching hypothesis is that enhanced STAT3 signaling, TGF? imprinting, and IDO/TDO resistance in NK cells will potentiate adoptive immunotherapy against pediatric cancers. We will test these hypotheses by determining whether i) deletion of the SOCS3 locus and prolonged STAT3 signaling will drive intratumoral antigen-specific proliferation, function, and survival, ii) TGF? imprinting in NK cells will enhance effector function and adaptive crosstalk, and iii) AHR deletion will promote effector function, and adaptive crosstalk, all of which enhance the pro-inflammatory TME for improved anti-tumor responses. We will use xenograft sarcoma models to investigate the mechanisms underlying these interactions and test therapeutic strategies for translation to the clinic. Using T-cell replete models, high-dimension phenotyping, and immunogenomic approaches we will assess cross-talk with adaptive immunity to better understand the broader immune consequences on the inflammatory microenvironment. Through this work we seek to overcome the hurdles of low tumor antigenicity and immunosuppression necessary for advancing immunotherapy against solid tumors. If we are successful, our results will support translation into clinical trials for pediatric cancers.